TY - JOUR
T1 - Cdc42 and PI(4,5)P2-induced actin assembly in xenopus egg extracts
AU - Lebensohn, Andres M.
AU - Ma, Le
AU - Ho, Hsin-Yi Henry
AU - Kirschner, Marc W.
PY - 2006
Y1 - 2006
N2 - Xenopus egg cytoplasmic extracts have been used to study a variety of complex cellular processes. Given their amenability to biochemical manipulation and physiological balance of regulatory proteins, these extracts are an ideal system to dissect signal transduction pathways leading to actin assembly. We have developed methods to study Cdc42 and PI(4,5)P2-induced actin assembly in Xenopus egg extracts. In this chapter, we describe detailed procedures to prepare Xenopus egg extracts, Cdc42, and PI(4,5)P2 for use in actin assembly experiments. We also describe a fluorometric pyrene actin assay for quantitative kinetic analysis of actin polymerization and a microscopic rhodamine actin assay for quick measurement of actin rearrangements in extracts. Finally we provide a protocol for immunodepletion of proteins and discuss the use of immunodepletion and rescue experiments for functional analysis of components in the extracts.
AB - Xenopus egg cytoplasmic extracts have been used to study a variety of complex cellular processes. Given their amenability to biochemical manipulation and physiological balance of regulatory proteins, these extracts are an ideal system to dissect signal transduction pathways leading to actin assembly. We have developed methods to study Cdc42 and PI(4,5)P2-induced actin assembly in Xenopus egg extracts. In this chapter, we describe detailed procedures to prepare Xenopus egg extracts, Cdc42, and PI(4,5)P2 for use in actin assembly experiments. We also describe a fluorometric pyrene actin assay for quantitative kinetic analysis of actin polymerization and a microscopic rhodamine actin assay for quick measurement of actin rearrangements in extracts. Finally we provide a protocol for immunodepletion of proteins and discuss the use of immunodepletion and rescue experiments for functional analysis of components in the extracts.
UR - http://www.scopus.com/inward/record.url?scp=32144433330&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=32144433330&partnerID=8YFLogxK
U2 - 10.1016/S0076-6879(06)06013-7
DO - 10.1016/S0076-6879(06)06013-7
M3 - Article
C2 - 16472657
AN - SCOPUS:32144433330
VL - 406
SP - 156
EP - 173
JO - Methods
JF - Methods
SN - 0076-6879
M1 - 13
ER -